Amphoteric, isoelectric immobiline membranes for preparative isoelectric focusing

Journal of Biochemical and Biophysical Methods, 14 (1987) 29-43 Elsevier

29

BBM 00581

Amphoteric, isoelectric Immobiline membranes for preparative isoelectric focusing Pierre Wenger ~, Maxime de Zuanni t, Philippe Javet 1, Cecilia Gelfi 2 and Pier Giorgio Righetti 2 I Swiss Federal Institute of Technology, Institute of Chemical Engineering, Lausanne, Switzerland, and ' Department of Biomedical Sciences and TechnologT, Unit,ersity of Milan, Milan, haly

(Received 29 September 1986) (Accepted 17 November 1986)

Summary Amphoteric, isoelectric agarose membranes, as devised by Martin and Hampson [Martin, A.J.P. and Hampson, F. (1978) J. Chromatogr. 159, 101-110], are found unsuitable for blocking electrocndosmosis in multi-compartment eleetrolysers during preparative isoelectric focusing, due to the poor and highly unpredictable incorporation of carboxyls and amino groups on the polysaccharide moiety. New, polyacrylamide-based membranes are described, containing as buffers and titrants the Immobiline chemicals used to produce immobilized pH gradients. These new membranes are supported on both faces by a non-woven polypropylene cloth, a material exhibiting minimal adsorption properties for proteins. Due to the extensively developed Immobiline technology, membranes with highly predictable isoelectric points, well-defined buffering capacity and conductivity can be synthesized at any pH value along the pH 3-10 scale. They are effective in blocking electroendosmosis even when the ApH on either side of the membrane is as high as 1.5 pH unit. Key words: Preparative isoelectric focusing: Immobilized pH gradient; Amphoteric membrane: Multicompartment electrolyzer.

Introduction In 1978, Rilbe [1] and Martin and Hampson [2] published independently and simultaneously a new concept for large-scale preparative isoelectric focusing (IEF), termed 'steady-state rheoelectrolysis'. It consisted in creating a pH gradient by Correspondence address: Prof. P.G. Righetti, University of Milan, Via Celoria 2, Milano 20133, Italy. 0165-022X/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

30 using simple, non-amphoteric buffers (e.g. acetic acid/sodium acetate for a pH 4-5 gradient) instead of the costly, carrier ampholyte buffers commonly used in standard IEF [3]. These non-amphoteric buffers have several advantages, such as low cost, high ionic strength and well defined conductivity and buffering capacity, which appear promising for large-scale protein separations. Different groups [1,2,4] have studied the stabilization of the pH profile generated by electrolysis of simple, non-amphoteric buffers in free solution. In one approach [1], the loss of buffering ions and titrants from the electrophoretic chamber towards the opposite electrodes is compensated for by an external, hydraulic flow matching, in principle, the electrophoretic ion depletion. In another approach [2], the diminution or accumulation of ions in the end compartments are compensated for by external additions from large electrode reservoirs, without recycling as described [1]. As these separations are in general performed in free solution, stabilization of the bulk liquid against convection is obtained by designing multi-compartment electrolyzers [5], divided by electrically permeable membranes. These membranes, in reality, have generated more problems than they could solve: they are in general electrically neutral, but acquire charges by adsorption of ions from the electrolytes and by trapping large protein molecules during the electrophoretic process. This generates electroendosmosis, a term denoting bulk liquid flow through the membrane caused by the presence or acquisition of a net electrical charge. To overcome this problem, in 1981 Martin and Hampson [6] patented the idea of amphoteric, isoelectric membranes having a good buffering capacity at their isoelectric point. In virtue of their buffering power, these membranes would remain under stable, substantially isoelectric conditions during the electrophoretic process, and therefore they would tend to effectively block any incipient electroosmotic flow. These membranes would ideally possess a high density of charges of both signs, imparting to them a high buffering capacity at the isoelectric point, so that any potential ion adsorption would cause negligible effects. As an ideal membrane, Martin and Hampson [6] proposed a cloth-supported agarose layer, carboxylated with chloroacetic acid to introduce -CH2COOH groups and subsequently amino alkylated with diethylamino ethyl halides to link -CH2CH2NEt 2 counterions. In a series of papers, we have recently: (a) redefined the mathematical principles ensuring stable, steady-state concentration profiles in non-amphoteric buffer focusing [7]; (b) built an improved multicompartment apparatus fulfilling the above requirements [8] and (c) demonstrated its potential in the separation of model mixtures of amino acids [9]. However, upon prolonged experience, we found the amphoteric agarose membranes [6] to give unsatisfactory results. Basically, their synthesis was not very reproducible and they exhibited a gap of 4 pH units where they could not buffer nor have predictable pl values (see the data in the present report). With the advent of immobilized pH gradients (IPG) [10,11] we realized that we had the right tool for creating amphoteric, isoelectric membranes of fully controlled conductivity, buffering capacity and pI values, amenable to highly reproducible synthetic processes. This report deals with the synthesis of such lmmobiline-based membranes, supported by a hydrophylic polyacrylamide network, and with their physico-chemical properties.

31 Materials and Methods Among the many commercial tissues available, we have selected a non-woven polypropylene cloth (Paratherm PP 330-40, Jenos SA, Lausanne, Switzerland) as a support for our agarose or polyacrylamide membranes, as it exhibited minimal protein adsorption. Agarose IEF and Immobilines (pK values of 4.6, 6.2, 7.0 and 9.3) were from LKB Produkter AB, Bromma, Sweden. Acrylamide, N, N'-methylene bisacrylamide, persulphate and T E M E D were from Bio Rad, Richmond, CA, U.S.A.

Measurements of electroendosmosis For that, we have built the apparatus drawn in Fig. 1, with a membrane-supporting disk depicted in Fig. lb. The cell consists of two chambers (A) of about 200 ml volume. These chambers are filled with a wide-spectrum buffer, consisting of equivalent concentrations of acetic acid (2.9 × 10-3 M), phosphoric and boric acids, titrated to any desired p H with 1 N N a O H . Such mixture can cover a p H 3-10 spectrum. The amphoteric, isoelectric membrane (M), supported by the disk of Fig. lb, is clamped between the two chambers (A), the O-ring ensuring a flow-tight connection. The two electrode compartments (E), contain two nickel electrodes of about 25 cm z of surface (Ni wire of 2 m m diameter wound on a spiral). The anolyte is a saturated solution of potassium ferrocyanide while the catholyte is a saturated solution of potassium ferricyanide. Both solutions are gently overlayed with a viscous layer of polyethylene glycol (PEG) to quench diffusion and avoid mixing between electrolyte and buffer solutions in the two chambers (A). The viscous PEG

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32 layer and the electrolytes contain additionally 0.2 M sodium sulphate, to ensure good conductivity. A potential difference of 315 V with up to 20-30 mA can be applied without gas bubbling from the electrodes. The two chambers (A) can be emptied from two valves (R). Two thermometers (T), in line with the electrode wires, ensure adequate temperature monitoring. Two capillaries (C) of 1.2 mm diameter allow accurate measurements of water transport ( # l / m i n ) from one chamber to the other (electroendoosmosis). As a convention, we have adopted a positive sign for a flow from the cathodic to the anodic compartment and a negative sign for the opposite flow.

Measurement of the isoelectric point of amphoteric membranes The cell (Fig. 1) is filled with a buffer solution of a given pH. A 315 V potential difference is applied for 15 min and every minute a reading is taken of the water flow from one chamber to the other (A) by observing the meniscus displacement in the capillaries (C). The average osmotic flow (Veo) at a given pH is thus obtained. By repeating the measurements at different pH values, it is possible to plot the profile of electroosmotic flow as a function of pH (see, as an example, the panels of Fig. 4). The isoelectric point ( p I ) of the membrane will be the pH at the cross-over point, i.e. the pH at which Iieo = O.

Synthesis of amphoteric, agarose membranes The gelling chamber consists of two aluminum walls of 295 x 200 x 15 mm containing cooling (or heating) serpentines. The spacers are steel gaskets of variable thickness (in general, 1-3 mm). The chamber thus formed is assembled beneath a hydraulic press. Before closing the chamber, two pieces of cloth are wetted and stretched against the two aluminum walls. The chamber is heated to 70°C by circulating hot water through the serpentines and then filled with hot (80°C) 4% agarose solution, poured in the gap between the two cotton tissues. The heated chamber, clamped in the hydraulic press at 1 atm of pressure, is now cooled by circulating water at 10°C. After 10 min, the agarose membrane (supported on both sides by the cotton tissue) is rendered amphoteric by the method of Martin and Hampson [2]. It is first incubated 2 h in 2.5 M N a O H and then overnight in 1% ( v / v ) epichlorohydrine in xylene. After an additional treatment for 2 h in 2.5 M NaOH, the membrane is subsequently incubated for 2 h in 1 M monochloroacetic acid dissolved in 2.5 M NaOH. After blotting with filter paper, the agarose membrane is bathed for 15 min in 2.5 M N a O H containing variable amounts of diethanolamine (in general between 0.06 and 0.12 M) according to the desired p l to be obtained for a given membrane. The now amphoteric membrane is hardened overnight in 8% epichlorohydrine in xylene. After rinsing in distilled water, the membrane is stored in a sodium fluoride solution (500 mg/l).

Synthesis of amphoteric lmmobiline membranes A transversal section of the chamber in which the membranes are polymerized is shown in Fig. 2. The outer walls of the chamber are two 3-ram thick glass slabs. They are separated by gaskets of variable thickness (in general, 1-3 mm). A second,

33

glaSS plates

Fig. 2. Transversal section of the chamber for polymerizing amphotefic, Immobiline membranes. Note the 0.2 mm thinner gasket for supporting the two layers of polypropylene cloth.

inner gasket is placed in the chamber, 0.2 mm thinner than the outer spacer. On this inner gasket are resting the two stretched porous polypropylene membranes acting as outer supports for the Immobiline matrix. The inner gap is filled with a solution of monomers (in general 10 or 15% T, with a fixed cross-link value of 3% C) containing variable amounts of buffering and titrant Immobilines in the ratios needed to generate any desired isoelectric point [11]. We have tried any possible range of Immobiline molarities, from a minimum of 10 mM up to 100 mM buffering ion. The Immobiline membrane is now polymerized, around neutral pH, at 50°C in a forced-ventilation oven for 1 h [12]: these polymerization conditions are an absolute requirement, as only at 50°C the Immobiline chemicals are incorporated with high efficiency (ca. 85-90% conversion) and at a ratio buffering/titrant approximating unity, thus ensuring highly reproducible isoelectric points.

Results

Properties of amphoteric, agarose membranes Table 1 gives the properties of two amphoteric agarose membranes, one prepared with 0.12 M, the other with 0.06 M diethanolamine. It can be seen that they are indeed efficient in blocking and reversing the electroosmotic flow according to the prevailing pH in the solution bathing the membrane, and that they exhibit an isoelectric point (pl; last column to the fight). However, one problem is immediately apparent: the synthetic process is quite unreproducible, yielding membranes of widely different p l values from identical preparations (in the lower part of the table, two duplicate membranes gave a p l = 5.38 and a p l = 4.85, respectively). A second, severe problem is revealed by Fig. 3: by assuming that the carboxyls grafted to the agarose have an average p K = 4.6 and that the basic counterions have a pK = 9.3, it is possible to simulate the profiles of the pH gradients (solid curve), degree of ionization (a, broken lines) and buffering power (dotted gaussians) along

34

TABLE 1 ELECTROOSMOTIC FLOW (V~o)and ISOELECTRIC POINTS (pl) OF AMPHOTERIC AGAROSE MEMBRANES '~ Membrane

Solution pH

+ 0.12 M diethanolamine I 2.65 6.1 7.1 8.0 II

3.1 3.1 6.2 6.2 8.0 9.0

+ 0.06 M diethanolamine I 3.0 5.0 6.0 II

4.0 5.0 6.0

Ve,, [M/min]

I [mA]

U IV]

T [°C1

Measured p/

12.4 4.6 1.3 - 1.1

3(1 27 16 30

300 315 30() 285

28 28 28 28

7,7

21 19.3 7.3 5.5 1.3 - 2.6

18 18 21 18 18 18

315 315 315 240 200 165

26 28 28 30 26 27

8.0

10.1 1.1 -2.3

16 16 18

315 315 315

25 26 26

5.38

2.5 ~0 - 3.5

12 16 20

315 315 315

28 28 27

4.85

All membranes treated with 1 M C1-CHzCOOH in 2.5 M NaOH and then the upper group with 0.12 M and the lower group with 0.06 M diethanolamine.

the p H scale, by a c o m p u t e r p r o g r a m similar to the one of Dossi et al. [13] for immobilized p H gradients. It is seen that such agarose m e m b r a n e s could, at most, buffer properly only in the p H 4 - 5 a n d p H 9 - 1 0 regions, while in the p H 5 - 9 span (where most proteins are isoelectric) they would be totally useless, as they are m a x i m a l l y ionized ( a a p p r o a c h i n g unity) a n d m i n i m a l l y buffering. This also m e a n s that, in the p H 5 - 9 interval, any slight error in grafting the molar fraction of P (the basic titrant) results in huge variations of the a p p a r e n t isoelectric p o i n t of the agarose m e m b r a n e s . Thus, the data of T a b l e 1 and the simulations of Fig. 3 show that essentially agarose m e m b r a n e s are not a useful approach to resolving the p r o b l e m of electroendosmosis in m u l t i c o m p a r t m e n t electrolyzers. They were thus a b a n d o n e d in favour of I m m o b i l i n e m e m b r a n e s .

Properties of amphoteric Immobiline membranes Fig. 4 gives the properties of four different I m m o b i l i n e m e m b r a n e s , all based o n a polyacrylamide matrix of 10% T, 3% C composition. In each panel, the lower left corner reports the molarities a n d p K values of the two I m m o b i l i n e s used to generate each m e m b r a n e ; below it, the theoretical p l value is reported. It should be noted that the p l of any l m m o b i l i n e m e m b r a n e can be predicted with high accuracy

35

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whereas this is not so for agarose membranes, cf. Table 1. A measured p l (Plm) can be interpolated from the curves of electroosmotic flow along the pH scale. As seen in Fig. 4, the theoretical and experimentally measured pI values are in excellent agreement, rarely diverging by more than 0.1 pH unit. Table 2 summarizes the data of these four membranes by comparing their theoretical and experimental p l values. In the case of the p l = 5.40 membrane, we have prepared four types, in order to check the reproducibility: a duplicate was made to incorporate 10 mM each of pK 4.6 and 6.2 Immobilines, while a second set contained 40 mM each of the two species. As seen from the measured p l values, the reproducibility of preparation of these membranes is impressive (given the highly developed know-how in the IPG field, this was simply expected). We have next addressed the question of the proper levels of Immobilines to be grafted in the polyacrylamide membrane. We have thus taken the p l = 5.40 membrane and prepared a 10 mM (Fig. 5A), a 40 mM (Fig. 5B) and a 100 mM (Fig. 5C) surface. While the 10 and 40 mM membranes exhibit correct electroosmotic properties and yield accurate experimental p l values, the 100 mM surface exhibits much larger dispersion and shows anomalous flow profiles in the pH range surrounding the pI. It seems reasonable thus to set an upper molarity limit of about 50 mM of each Immobiline in the membrane. The physico-chemical properties of the mem-

36

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TABLE 2 THEORETICAL A N D EXPERIMENTAL p l VALUES OF AMPHOTERIC, IMMOBILINE-POLYA C R Y L A M I D E MEMBRANES a Immobiline molarities p K 4.6

p K 6.2

5×10 4 M 1×10-2M 1 × 1 0 -2 M 4 × 1 0 ..2 M 4g10 2 M

1.5×10-3 M 1 ×10 2 M 1 ×10 .2 M 4 xl0 2 M 4 ×10-2 M

p K 4.6

p K 7.0

2×10 2 M 6×10-2 M

6 ×10-2 M 2 ×10-2 M

Theoretical pI

Experimental pI

6.50 5.40 5.40 5.40 5.40

6.55 5.46 5.4,4 5.42 5.25

7.30 4.30

7.24 4.27

a All membranes made with a 10'~ T, 3~ C polyacrylarmde matrix

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38

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Fig. 6. Physico-chemical properties of some Immobiline membranes. (A) Titration of a polyacrylamide membrane containing Immobilines pK 4.6 and 6.2 in the pH 4-7 range. (B) Titration of a polyacrylamide membrane containing Immobilines pK 4.6 and 7.0 in the pH 4-8 interval Note that even in the last, quite unfavourable case, good buffering power (g) and degree of ionization (a) profiles are obtained all along the titration interval. Computer simulations as described [13]

39

branes listed in Table 2 are shown in Fig. 6A (for the couple pK values 4.6 and 6.2) and in Fig. 6B (for the couple pK values 4.6 and 7.0). As judged from the smooth pl m profiles in both panels, essentially at any given ratio buffering/titrant these membranes ensure highly predictable pI values (in the pH 4-7 region in Fig. 6A, in the pH 4-8 interval in Fig. 6B). Even in the last case, half-way through the titration (molar fraction of P = 0.5; theoretical pl = 5.8) there is still enough buffeting power to ensure a proper behaviour of the membrane (compare the present buffeting power curve with that of Fig. 3). Behaviour of the Immobiline membranes in a A p H environment

The above studies are indeed more theoretical than practical, as they show the behaviour of agarose or polyacrylamide membranes when bathed in solutions having different pH values from the pI of the membrane, but of equal values in

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Fig. 7. Behaviour of Immobiline membranes in a ,.ApH environment. In the three cases, the same Immobiline membrane has been used, with a theoretical p l = 4.16. (a) The pH of the cathodic side is kept constant at 4.51, while on the anodic side it is progressively lowered to pH 3.4; (b) cathodic pH = 4.76, anodic pH from 4.76 to 3.50; (c) cathodic pH = 4.99, anodic pH from 4.99 to 3.5. Note that even in a wide A pH (1.2) environment the Immobiline membranes can effectively stop electroendoosmosis.

40 TABLE 3 BEHAVIOUR p H -.b

4.99 4.99 4.99 4.99 4.99

OF IMMOBILINE

p H ''~

4.99 4.50 4.17 3.76 3.55

pH

4.99 4.75 4.58 4.38 4.27

MEMBRANES - log C~l"

4.99 4.68 4.41 4.04 3.84

IN A ApH ENVIRONMENT

a

M e a s u r e d V,o

C a l c u l a t e d Veo ( ~ l / m i n )

(~al/min)

pH

- log C H "

- 5.0 - 3.6 - 2.6 + 0.3 + 1.4

- 5.2 - 4.0 2.7 - 1.4 - 0.4

* +

5.2 3.4 1.7 0.7 1.6

a p l 4.16 I m m o b i l i n e m e m b r a n e w a s used. R e f e r to c u r v e (c) in Fig. 7. b p H in the c a t h o d i c c o m p a r t m e n t . p H in the a n o d i c c o m p a r t m e n t .

both sides. In a multicompartment electrolyzer, in presence of a pH gradient, the membrane Will be confronted with solutions of unequal p H in either side. How effective will such a surface be in blocking electroendosmosis? To answer this problem, we have performed the experiment of Fig. 7: a membrane having a theoretical p I = 4.16 was bathed in solutions of progressively greater A p H values (by that we define the difference in p H between the cathodic and anodic sides of the membrane) on both sides. In the case of curve (a), the cathodic p H was fixed at 4.51 while on the anodic side it was progressively varied down to p H 3.4. In the case of curve (b), the cathodic p H was fixed at 4.76, while at the anode it was progressively lowered to p H 3.30. In the last case (curve c), the cathodic p H was set at 4.99, while the anodic p H was progressively lowered to p H 3.5. As shown in Fig. 7, even under these substantially varying zapH conditions on the two faces of the membrane, the electroosmotic flow can be eliminated. As an example, the data for the experiment pertaining to curve (c) are shown in Table 3. Interestingly, however, the net electroosmotic flow does not become zero when the arithmetical mean (pH in Table 3) of the p H values of the two limiting buffer solutions on either side equals the isoelectric point of the membrane, but when the negative logarithm of the arithmetical mean of the hydrogen ion concentration ( - l o g (~rt'in Table 3) on either side matches the isoelectric point of the membrane (as seen in Table 3, the difference between these two values can be as high as 0.1 p H unit).

Discussion The mechanism by which an amphoteric, isoelectric membrane should block electroendosmosis during isoelectric focusing is exemplified by the following reasoning. Suppose that such a membrane is bathed, in an isoelectric focusing cell, between two solutions of different pH, whose arithmetic mean is equal to the p I of the membrane. Such a membrane will thus bear no net charge and should not generate any appreciable electroendoosmotic flow. If now such a surface adsorbs

41 positive charges (e.g. from the buffer components or by protein trapping), an anodic electroosmotic flow will be generated, forcing solutions having pH > pl to cross the membrane. As a result, negative charges will be generated on the surface, which will counterbalance the initial positive charge acquired by the membrane. The electroosmotic flow will now cease. This basic principle, which underlies the Martin and Hampson patent, has been fully supported and verified by our data. What we have found quite unsatisfactory, however, is the choice of the membrane: agarose surfaces, rendered isoelectric by grafting -CH 2COOH and -CH 2CH 2NEt z counterions, appear to have severe defects. First of all, the grafting process cannot be properly controlled, resulting in membranes of substantially varying pl values from identical preparations. Secondly, due to lack of a variety of buffering groups with appropriate pK values, a gap of 4 pH units (pH 5-9) exists, where no suitable amphoteric agarose membranes can be expected to be ever produced. These problems are completely solved by using the know-how and technologies developed for IEF in immobilized pH gradients (IPG). IPGs are created with the aid of seven different buffers, by the commercial name of Immobiline, having pK values quite evenly distributed along the pH axis (the pK values 3.6, 4.4 and 4.6 lmmobilines contain weak carboxyls, while pK values 6.2, 7.0, 8.5 and 9.3 consist of tertiary amino groups). As these buffering ions are grafted to the nitrogen of the amido group of acrylamide, they exhibit reactivities quite similar to those of the basic monomers of the polyacrylamide matrix, i.e. acrylamide and the cross-linker bisacrylamide. It should be noted that, as the starting solution contains only monomers with acrylic-type double bonds, the Immobilines are effectively grafted onto the growing polymer chain with a stochastic distribution. This is not guaranteed by the Martin and Hampson procedure, where buffering ions and titrants have to be linked to a pre-existing polysaccharide coil. Due to the fact that, in gelled agarose, the strands are arranged in a double helix, which is then further laterally aggregated in bundles of 9-11 helixes to form a rigid, pillar-like structure [14], it is predictable that grafting reactions could have low efficiencies. Conversely, Immobiline-based polyacrylamide membranes can be produced with a high degree of reproducibility and are expected to exhibit very precise isoelectric points with any desired degree of buffering capacity, conductivity and ionic strength built on the surface. In addition, the simple fact of having seven pK values well distributed along the pH axis (six in reality, as the pK 4.4 and 4.6 Immobilines are too close and should not in general be used simultaneously) allows the production of an extremely large variety of p! values, also distributed at any desired point along the pH 3-10 interval. The synthesis of such membranes is quite simple: once selected a buffering ion (in a 10-50 mM concentration) addition of a given molar fraction of titrant (see Fig. 6), as defined by the Henderson-Hasselbalch equation, will automatically ensure production of a membrane of a given pI. As long as this buffering ion is titrated in a pH interval of _ 0.5 pH unit about its pK value, the membrane thus generated will possess good buffering power and proper ionic strength. It should in fact be noted that such membranes are indeed synthesized on a principle analogous to that of 'good' carrier ampholytes, i.e. with the requirement of closely spaced pK values, as specified by Rilbe [15] in the early development of isoelectric focusing.

42

Experimentally, several questions remain still unanswered. E.g., what is the relative importance of the buffering power of the membrane vs. that of the surrounding solutions? Is it preferable to increase or to diminish the buffering capacity of a given membrane? According to Martin and Hampson [6], the latter solution is preferable, but this is still to be verified. How does the electroosmotic flow change when varying the buffer constituents surrounding the membrane? How porous can a polyacrylamide membrane be made to allow for good protein cross-migration while retaining proper mechanical strength and flow tightness? Would it be advisable to change altogether the type of membrane? E.g., given the fact that cellulose acetate membranes are extensively used and are readily available, it would be desirable to graft the Immobiline chemicals to such supports. Work is in progress to elucidate all these aspects.

Simplified description of the method New amphoteric, isoelcctric membranes, to bc used in multicompartment electrolygers for preparative isoelectric focusing, are described, based on the chemistry and know-how of immobilized pH gradients. By using any of the seven, non-amphoteric lmmobiline chemicals, at proper buffer/titrant ratios (as defined by the Henderson-Hasselbalch equation) any amphoteric membrane, of well defined isoclectric point, buffering capacity, conductivity and ionic strength, can be synthesized in the pH 3-10 interval. These membranes are sandwiched between two layers of non-woven polypropylene. They are effective in blocking electroendoosmotic flow in multicompartment electrolyzers even at pH values quite removed from the membrane p l (up to a ApH = 1.5 pH unit).

Acknowledgements P.G.R. is supported by grants from Consiglio Nazionale delle Ricerche (CNR, Roma), Progetti Finalizzati 'Chimica Fine a Secondaria' e 'Biotecnologie'. P.W. has been supported by fellowships from the Swiss Institute of Technology of Lausanne and of the University of Milan for a stay at the University of Milan for this cooperative project.

References 1 2 3 4 5 6 7 8 9

Rilbe, H. (1978) J. Chromatogr. 159, 193-205 Martin, A.J.P. and Hampson, F. (1978) J. Chromatogr. 159, 101-110 Svensson, H. (1961) Acta Chem. Scand. 15, 325-341 Bier, M., Mosher, R.A., Thormann, W. and Graham, A. (1984) in Electrophoresis '83 (Hirai, H., ed.), de Gruyter, Berlin, pp. 99-107 Rilbe, H., Forchheimer, A., Petterson, S. and Jonsson, M. (1975) in Progress in lsoelectric Focusing and Isotachophoresis (Righeni, P.G., ed.), Elsevier, Amsterdam, pp. 51-63 Martin, A.J.P. and Hampson, F. (1981) U.S. Patent No. 4.243.507 Wenger, P. and Javet, Ph. (1986) J. Biochem. Biophys. Methods 13. 259-273 Wenger. P. and Javet, Ph. (1986) J. Biochem. Biophys. Methods 13,275-287 Wenger, P. and Javet, Ph. (1986) J. Biochem Biophys. Methods 13,289-303

43 10 BjeUqvist, B., Ek, K., Righetti, P.G., Gianazza, E., G6rg, A., Westermeier, R. and Postel, W. (1982) J. Biochem Biophys. Methods 6, 317-339 11 Righetti. P.G. (1984) J. Chromatogr. 300, 165-223 12 Righetti, P.G., Ek, K. and Bjellqvist, B. (1984) J. Chromatogr. 291, 31-42 13 Dossi, G.. Celentano, F., Gianazza, E. and Righetti, P.G. (1983) J. Biochem. Biophys. Methods 8, 109-133 14 Arnott, S., Fulmer, A., Scott, WE., Dea, I.C.M., Moorhouse, R. and Rees, D.A. (19741 J. Mol. Biol. 90, 269-284 15 Rilbe, H. (1976) in Isoelectric Focusing (Catsimpoolas, N., ed.) Academic Press, New York, pp. 14-52